The synthesis and biological evaluation of a series of new derivatives of 2-substituted 5-phenyl-
1,4-benzodiazepines, structurally related to tifluadom (5), are reported. Chemical and
pharmacological studies on compounds 6 have been pursued with the aim of expanding the
SAR data and validating the previously proposed model of interaction of this class of compounds
with the -opioid receptor. The synthesis of the previously described compounds 6 has been
reinvestigated in order to obtain a more direct synthetic procedure. To study the relationship
between the stereochemistry and the receptor binding affinity, compounds 6e and 6k were
selected on the basis of their evident structural resemblance to tifluadom. Since a different
specificity of action could be expected for the enantiomers of 6e and 6k, owing to the results
shown by (S)- and (R)-tifluadom, their racemic mixtures have been resolved by means of liquid
chromatography with chiral stationary phases (CSP), and the absolute configuration of the
enantiomers has been studied by circular dichroism (CD) and 1H NMR techniques. Moreover,
some new 2-[(acylamino)ethyl]-1,4-benzodiazepine derivatives, 6a-d,f,g,j, have been synthesized,
while the whole series (6a-o) has been tested for its potential affinity toward human
cloned -opioid receptor. The most impressive result obtained from the binding studies lies in
the fact that this series of 2-[2-(acylamino)ethyl]-1,4-benzodiazepine derivatives binds the
human cloned -opioid receptor subtype very tightly. Indeed, almost all the ligands within
this class show subnanomolar Ki values, and the least potent compound 6o shows, in any case,
an affinity in the nanomolar range. A comparison of the affinities obtained in human cloned
-receptor with the correspondent one obtained in native guinea pig -receptor suggests that
the human cloned -receptor is less effective in discriminating the substitution pattern than
the native guinea pig -receptor. Furthermore, the results obtained are discussed with respect
to the interaction with the homology model of the human -opioid receptor, built on the recently
solved crystal structure of rhodopsin. Finally, the potential antinociceptive and antiamnesic
properties of compounds 6e and 6i have been investigated by means of the hot-plate and passive
avoidance test in mice, respectively.

The synthesis and biological evaluation of a series of new derivatives of 2-substituted 5-phenyl-
1,4-benzodiazepines, structurally related to tifluadom (5), are reported. Chemical and
pharmacological studies on compounds 6 have been pursued with the aim of expanding the
SAR data and validating the previously proposed model of interaction of this class of compounds
with the -opioid receptor. The synthesis of the previously described compounds 6 has been
reinvestigated in order to obtain a more direct synthetic procedure. To study the relationship
between the stereochemistry and the receptor binding affinity, compounds 6e and 6k were
selected on the basis of their evident structural resemblance to tifluadom. Since a different
specificity of action could be expected for the enantiomers of 6e and 6k, owing to the results
shown by (S)- and (R)-tifluadom, their racemic mixtures have been resolved by means of liquid
chromatography with chiral stationary phases (CSP), and the absolute configuration of the
enantiomers has been studied by circular dichroism (CD) and 1H NMR techniques. Moreover,
some new 2-[(acylamino)ethyl]-1,4-benzodiazepine derivatives, 6a-d,f,g,j, have been synthesized,
while the whole series (6a-o) has been tested for its potential affinity toward human
cloned -opioid receptor. The most impressive result obtained from the binding studies lies in
the fact that this series of 2-[2-(acylamino)ethyl]-1,4-benzodiazepine derivatives binds the
human cloned -opioid receptor subtype very tightly. Indeed, almost all the ligands within
this class show subnanomolar Ki values, and the least potent compound 6o shows, in any case,
an affinity in the nanomolar range. A comparison of the affinities obtained in human cloned
-receptor with the correspondent one obtained in native guinea pig -receptor suggests that
the human cloned -receptor is less effective in discriminating the substitution pattern than
the native guinea pig -receptor. Furthermore, the results obtained are discussed with respect
to the interaction with the homology model of the human -opioid receptor, built on the recently
solved crystal structure of rhodopsin. Finally, the potential antinociceptive and antiamnesic
properties of compounds 6e and 6i have been investigated by means of the hot-plate and passive
avoidance test in mice, respectively.